Recipient Organization
DRY CREEK LABORATORY
1618 BALDWIN RD.
HUGHSON,CA 95326
Performing Department
(N/A)
Non Technical Summary
Crown gall is a multimillion dollar worldwide problem in fruit and nut orchards, vineyards and nurseries. Agrobacterium tumefaciens, a ubiquitous soil bacterium, causes the formation of galls on plant stems which eventually lead to reduced yields and eventually destruction and/or replacement of the plant. Treatment options are limited and often ineffective. The purpose of this project is to produce transgenic commercial apple rootstocks which are resistant to crown gall. Phase I of this project produced transgenic fruit tree rootstocks that suppress crown gall. Phase II will produce crown gall resistant apple rootstocks that have commercial value, further characterize the transgenic events that provide resistance, and investigate methods for increasing the durability of crown gall resistance.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
1. The effectiveness of hairpin RNA (inverted repeat) constructs that target iaaM will be compared to the opposing-promoter constructs which conferred crown gall resistance to 12% of the transgenic apple trees produced during phase I studies. Although not critical, a higher success rate will aid in the introduction of the gall-resistance trait into a commercially useful apple rootstock. 2. To test whether protection is broad enough to be commercially useful, a variety of pathogenic Agrobacterium strains will be used to challenge apple lines that exhibit resistance to octopine-type A. tumefaciens. 3. Apple lines that silence A. tumefaciens oncogenes and prevent gall formation will be examined for transgene expression, copy number, and structure. 4. Intron-containing constructs that encode hairpin iaaH will also be tested for their ability to confer crown gall resistance. Transgenic plants that silence both iaaM and iaaH may have more durable gall resistance than lines
that silence only one of these oncogenes.
Project Methods
Inverted repeat constructs that contain iaaM sequences separated by an intron have been built by Dr. Ream's group. We will test these constructs in a commercial apple rootstock (M9) using procedures for production and testing of transgenic apple lines that were established in our laboratory during Phase I of this project. During Phase II we will generate at least 50 transgenic lines for each construct that will be tested, and then inoculate at least 180 root explants from each transgenic line with wild-type A. tumefaciens (octopine-type strain A348). Roots from nontransgenic plants and from lines transformed with vector sequences that do not confer resistance will be used as controls. To test whether protection is broad enough to be commercially useful we will use a variety of pathogenic A. tumefaciens strains to challenge root explants from transgenic lines that exhibit resistance to the initial challenge with strain A348. We will use seven pathogenic strains isolated
form apple as well as two extensively studied strains (C58 and Bo542). Dr. Ream's group will conduct molecular characterization of plants that show the strongest silencing of oncogenes. Initially, small amounts of tissue will be screened for the presence of transgene RNA by reverse transcriptase PCR. As larger quantities of tissue become available Northern blot analysis will be used to measure transgene mRNA and antisense RNA levels in transgenic root tissue. A hallmark of post-transcriptional gene silencing (PTGS) is strong transcription of target genes but little or no accumulation of mRNA. Nuclear runoff transcription assays will be conducted to asses transcription of transgenes in lines that do not accumulate transgene mRNA. Plants that exhibit gall resistance will be screened for short (21-23 nucleotide) antisense RNAs complementary to the PTGS-inducing transgene. Screening will be done by separating the small RNAs on denaturing polyacrylamide gels, transferring the RNAs to nylon
filters, and probing with strand-specific, radiolabeled RNAs which have been transcribed in vitro from iaaM coding sequences contained in a riboprobe vector. Because our transgenes contain the same 5' and 3' untranslated regions, these sequences will be excluded from transgene-specific probes. Signals will be detected by autoradiography and quantified using a phosphorimager. Transgene copy number, intactness and orientation will be examined by Southern analysis. Dr. Ream's laboratory will also construct a transgene that encodes a self-complimentary (hairpin) RNA containing inverted repeat iaaH sequences separated by an intron. The constructs will first be tested in roots of transgenic A. thaliana and in a mixed infection assay on Kalanchoe daigremontiana which will allow more rapid assessment of the effectiveness of the constructs. Efficient constructs will then be introduced into M9 apple rootstock. Initially the construct will be tested by itself in apple roots to determine its
effectiveness. Ultimately, we intend to introduce an iaaH silencing transgene into transgenic lines that have already demonstrated strong silencing of iaaM and further test the effectiveness of this stacked gene system.